1. Field of the Invention
Homocysteine (Hcy) is one in a series of intermediates produced along the transsulfuration pathway in which methionine is eventually converted to cysteine (Cys). The exclusive source of Hcy in mammals derives from the product of the enzyme catalyzed hydrolysis of S-adenosylhomocysteine. Once formed, Hcy may reenter the cycle through remethylation and conversion to methionine or combine with serine to form cystathionine, which is ultimately converted to Cys. The major metabolic pathway for the methylation of Hcy by methionine synthase requires vitamin B.sub.12 (cobalamine) as a methyl-transfer cofactor and 5-methyl-tetrahydrofolate as the ultimate methyl source. Not surprisingly, elevated levels of serum Hcy have been associated with insufficient intake of vitamin B.sub.12 or folate, or a deficiency in the ability to properly utilize these two vitamins. Moderately elevated levels of Hcy usually can be brought into balance by administering folate, a treatment for which there are few adverse side-effects.
In addition to anemia, individuals who are heterozygous for a defective cystathionine synthase gene, which results in a 50% diminution of normal enzymatic activity, are particularly susceptible to elevated Hcy levels following methionine loading. This genetic predisposition is the most common cause of moderate homocysteinuria (build-up of Hcy in urine) in otherwise healthy patients.
Lastly, there appears to be a correlation between moderately elevated levels of Hcy and cardiovascular disease.
For these reasons, there has been great interest in developing an accurate Hcy assay.
2. Description of the Related Art
There are several techniques to quantitate total homocysteine (Hcy) as well as distinguish between the free (reduced and disulfide) and protein-bound (primarily albumin) forms.
An excellent overview of the causes of homocysteinuria as well as an update on the current methods of clinical analysis can be found in Ueland, et al., Clin. Chem. 39(9):1764-1779 (1993).
An enzymatic method for a Hcy assay is described by Sundrehagen, et al., PCT/GB93/00138, where Hcy is assayed indirectly by measuring the product concentration following the enzyme catalyzed conversion of Hcy to S-adenosyl homocysteine.
High performance liquid chromatographic ("HPLC") methods for Hcy and Cys are known in the art. This analytical method discriminates between Hcy and Cys by differential adsorption and elution of the compounds on a chromatographic support. Andersson, et al., Clin. Chem. 39(8):1590-1597 (1993) describes the determination of total, free and reduced Hcy and Cys.
Hcy and Cys analysis by means of a gas chromatograph-mass spectrometer is described in Allen, et al., U.S. Pat. No. 4,940,658. Allen, et al., PCT/US92/05727 describes a chromatographic assay for cystathionine, the intermediary amino acid between Hcy and Cys produced in the metabolism of methionine.
Fiskerstrand, et al., Clin. Chem. 39(2):263-271 (1993) describes a fully automated analysis of total Hcy involving fluorescent labeling of serum thiols, followed by chromatographic separation of the Hcy derivative from the other sulfur-containing compounds.
Identification of Hcy by HPLC methods often involves derivatization with fluorescent reagents such as is described in Fiskerstrand, supra or a radioenzymatic technique such as is described in Refsum, et al., Clin. Chem. 31(4) 624-628 (1985). In addition, identification of Cys by protein sequence analysis involves derivatization with alkylating reagents. See, for example, Jue, et al., Analytical Biochemistry 210:39-44 (1993).
Unfortunately, chromatographic methods have the disadvantage of being slow and labor intensive. Furthermore, current methods of Hcy analysis require prior derivatization with fluorescent labels, such as bromobimane, in which the bromomethyl group reacts with the free thiol of Hcy, thus forming a thioether and releasing free bromide ion. The bromobimane reagent also reacts with all other free thiols in solution, therefore chromatographic separation of the various derivatized sulfur-containing species is necessary.
As many of the current methods of Hcy analysis rely on cumbersome chromatographic techniques, there is a need for a faster and simpler antibody-based assay for Hcy. To date, an immunoassay for Hcy has not been employed, based on the expectation that it would be problematic because the Hcy molecule is likely to be too small and contain too few antibody recognition features to serve as an effective hapten toward antibody development. Furthermore, antibodies may not attain the degree of antigenic specificity required to distinguish between Hcy and Cys, which differ in structure by a single methylene group. Accordingly, there is a present need for the development of an immunoassay method that would permit highly specific quantitation and differentiation of these compounds.
There are numerous techniques for handling undesirable cross-reactants. Brynes, et al., U.S. Pat. No. 4,952,336, describes a method of pretreating a sample with an aqueous periodate solution to eliminate cross-reactants in an amphetamine-methamphetamine immunoassay. Stevenson, PCT/GB90/01649, pertains to an improved immunoassay where the level of interference from rheumatoid factor is reduced by pretreating the sample with a reducing agent. Mach, et al., U.S. Pat. No. 4,978,632 pertains to an improved immunoassay where the level of interference from blood and blood products is eliminated by pretreating the sample with an oxidizing agent. These pretreatment methods only affect the cross-reactants; none of the methods affect the analyte.